黑曲霉pepB基因缺失菌株的构建及其功能分析

以黑曲霉(Aspergillus niger)GICC2773基因组DNA为模板，用PCR方法分别扩增pepB基因中的上游约1．4kb和下游约1．3kb两段DNA序列，将此两段序列按同一方向分别插入质粒pMW1中潮霉素抗性基因(hph)表达单元的5′和3′端，构建成重组质粒pMW1-pepB，用于通过同源重组靶向破坏基因组中的pepB基因。同源重组则采用原生质体-PEG方法，将酶切pMW1-pepB得到的线性片段转化A．niger GICC2773菌株，通过潮霉素选择平板得到62个Hgy抗性转化子，然后采用PCR方法从这些抗性转化子中筛选到1个由于同源重组产生的pepB基因缺失突变菌株pepB29。功能分析显示该突变株的酸性蛋白酶活性有明显下降，外源蛋白漆酶的分泌表达有所提高。

Construction and Functional Analysis of The pepB Gene Disruptant in Aspergillus niger

An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.